Functional analysis of a homeobox-containing gene expressed during early Xenopus laevis development

Xom is a homeobox-containing gene expressed during early stages of Xenopus laevis development which is involved in the specification of ventral tissues. Expression of Xom is induced as an immediate-early response to Bone Morphogenetic Protein-4, a member of the Transforming Growth Factor-β family. Moreover, Xom contains a novel homeodomain, which might affect its DNA binding specificity. This thesis describes a functional analysis of Xom. First, a preferred Xom DNA binding site was determined and the ability of Xom to bind potential binding sequences was tested in a series of in vitro assays. Together, these results showed that the sequence CTAATT(A/G) is critical for Xom to bind DNA, but that binding is greatly enhanced by the presence of an ATTA motif 6 or 7 nucleotides downstream of the core TAAT. A cell culture assay further demonstrated that Xom interacts with the selected sequence in vivo. Second, the ability of Xom to regulate transcription was analysed. Xom was shown to behave as a transcriptional repressor in Xenopus embryos and its repressing activity was mapped to N-terminal and C-terminal regions flanking the homeodomain. Xom's transcriptional repressing activity, together with its ventral expression pattern and ventralising activity in the early Xenopus embryo, suggested that Xom could function by down-regulating the expression of genes that are required for dorsal development in Xenopus. Consistent with this suggestion, over-expression of Xom RNA, or of a dominant-negative version, indicated that Xom regulates the expression of goosecoid, a homeobox-containing gene expressed in the organizer capable of partially mimicking the activity of the organizer. Finally, to test whether Xom acts by repressing goosecoid transcription directly, reporter constructs containing a goosecoid promoter fragment containing or lacking point mutations in potential Xom binding sites were co-injected with different effector RNAs into Xenopus embryos. These experiments suggested that at least part of the ability of Xom to repress goosecoid is direct, and identified a possible site to which Xom binds

Recruitment of RNA polymerase III to its target promoters

A key step in retrieving the information stored in the complex genomes of eukaryotes involves the identification of transcription units and, more specifically, the recognition of promoter sequences by RNA polymerase. In eukaryotes, the task of recognizing nuclear gene promoters and then transcribing the genes is divided among three highly related enzymes, RNA polymerases I, II, and III. Each of these RNA polymerases is dedicated to the transcription of specific sets of genes, and each depends on accessory factors, the so-called transcription factors, to recognize its cognate promoter sequences

Quantitative analysis of the effect of leflunomide on neural crest cell gene regulation during early embryonic development

The neural crest is a transient population of cells that arises at the border between the neural and non-neural ectoderm. These cells are induced, undergo an epithelial-to-mesenchymal transition, and then migrate along stereotypical pathways to form an array of derivatives such as pigment cells, cranio-facial cartilage and sensory neurons. Neural crest cells have long been studied and much about these cells and their interactions is still not fully understood. The small molecule compound leflunomide inhibits neural crest development. Leflunomide’s mode of action is to inhibit pyrimidine biosynthesis, thereby, preventing RNA transcription. Neural crest genes are actively transcribed and like many embryonic stem cells and tumour cells genes undergo an increased level of transcriptional pausing and subsequent elongation making a number of these genes sensitive to leflunomide. It was unclear at what stage of neural crest development leflunomide was acting. Here, I initially developed a quantitative approach using real-time PCR to measure gene expression in Xenopus. Secondly, using real-time PCR I have shown that neural plate border genes are not affected by leflunomide. Thirdly, the neural crest specification genes are affected and the pan neural plate marker Sox2 is not affected by leflunomide. I have confirmed by quantitative real-time PCR that the expression of genes involved in neural crest specification the proto-oncogene cMyc and cMyc responsive genes are affected. cMyc is implicated in embryonic stem cell transcriptional elongation and is well characterised to play an important role in neural crest specification

The YY1 transcription factor is a component of ribonucleoprotein complexes in xenopus laevis oocytes and embryos.

Yin Yang 1 (YY1) is a multifunctional transcription factor that is known mainly for its ability to activate or initiate transcription of a wide assortment of genes involved in cellular growth and differentiation. Xenopus laevis oocytes and embryos were used as a model to identify and characterize a potential developmental role for YY1. Northern and Western blots of oocyte and embryonic extracts showed YY1 mRNA and protein is expressed from the earliest stages of oocyte development through to tadpole stages. Examination of the transcriptional activity of YY1 in both oocytes and embryos using reporter gene constructs containing YY1-binding elements demonstrated that YY1 does not act as a repressor or activator of transcription either in oocytes or in embryos. Sub-cellular fractionation of oocytes and Western blot analysis showed YY1 is localized almost exclusively to the cytoplasm of oocytes and in cells of early embryos. Sequence analysis of YY1 revealed that it contains an established RNA binding motif located within the zinc fingers. A series of biochemical assays were performed to address the possibility that YY1 functions as a component of mRNPs in the oocyte cytoplasm. RNA gel mobility shift analyses using in vitro synthesized histone H2A transcripts and supershifts using YY1-specific antibodies suggested that YY1 or YY1-containing complexes in cytoplasmic extracts were able to bind RNA. Chromatographic analysis of oocyte lysates showed YY1 was specifically retained on oligo (dT) cellulose columns. Treatment of the same lysates with RNase abolished binding to oligo (dT), indicating that retention is dependent on the presence of intact polyadenylated RNAs. This suggested that YY1 may be a component of messenger ribonucleoprotein particles (mRNP). Separation of oocyte lysates by size exclusion chromatography (SEC) revealed that YY1 was present in large complexes with an approximate molecular mass of 480 kDa. RNase or phosphatase treatment of oocyte extracts released YY1 from high mass complexes. Analysis of phosphatase or RNase-treated extracts for DNA binding activity showed that monomeric YY1 was able to bind DNA with high affinity. Immunoprecipitation of YY1 complexes followed by cDNA synthesis and sequencing revealed that YY1 is associated with both ribosomal and messenger RNAs in the cytoplasm of the oocyte. These results indicate a novel function for YY1 as a component of messenger ribonucleoprotein particles

Atp-Dependent Chromatin Remodeling Complexes In Xenopus Development

Thesis (Ph.D.) University of Alaska Fairbanks, 2010A central question in the study of vertebrate development is how to account for the exquisite interplay of genes within differentiating cells and of groups of cells as they create the organs of the vertebrate embryo. Recently it has become clear that gene regulation by epigenetic processes adds a formerly unappreciated level of complexity to the regulatory network of development. One form of epigenetic gene regulation is embodied in ATP-dependent chromatin remodeling complexes, which use the energy of ATP hydrolysis to alter the interactions of DNA and histones. Chromatin remodeling complexes can both promote and repress expression of a gene at the appropriate time and place in vertebrate development. The list of their known roles in development is long and growing. Here I have studied the developmental role of CHRAC17, a subunit of the CHRAC and ATAC complexes, by visualizing its expression and by ablating CHRAC17 function in Xenopus laevis embryos. Whole mount in situ hybridization localized CHRAC17 expression to the neural tube, cranial placodes, and myotomes. Loss of CHRAC17 function following injection of embryos with CHRAC17-specific morpholino oligonucleotides resulted in abnormal development in the neural tube, eyes, notochord, and pharyngeal pouches, underlining the critical importance of CHRAC17 function in Xenopus development. Similarly, ablating the function of CHD4, the ATPase motor of the NuRD chromatin remodeling complex, resulted in severe developmental abnormalities in early Xenopus development

Proteolytic cleavage of cadherins: Functional role of the cleaved extracellular and cytoplasmic domains

Dynamic regulation of cadherin mediated cell-cell adhesion is crucial for morphogenesis and tissue homeostasis. Cadherin adhesive function can be regulated by distinct proteolytic cleavage events, resulting in release of either the ectodomain or cytoplasmic domain. However, it is unclear if the released fragments have biological activity by themselves. This thesis analyses the functional significance of the generated cadherin fragments. Using Xenopus laevis development as model system, it was shown in this thesis that the C-cadherin ectodomain has the capacity to interfere with morphogenetic movements in vivo, since overexpression of this fragment in early Xenopus embryos resulted in severe gastrulation defects. The observed defect was due to inhibition of convergent extension movements, a process that requires downregulation of cadherin adhesiveness. However, ectoderm integrity was not affected by expression of the cadherin ectodomain, demonstrating that this fragment specifically interferes with processes that require a tight regulation of cadherin function. The EC1 repeat of the cadherin extracellular domain contained all the necessary information to interrupt gastrulation movements. This activity was not dependent on the amino acid tryptophan at position 2, which is crucial for an adhesive functional molecule. Surprisingly, overexpressing the ectodomain of other classical cadherins caused similar defects, indicating the capacity for heterophilic interactions. The cadherin ectodomain may not only have a function during development, but could directly contribute to cancer progression. Indeed, a direct correlation was found between increased soluble E-cadherin levels in sera of melanoma patients and tumor mass. The cadherin cytoplasmic domain was shown to serve as a substrate for the PS1/gamma-secretase complex, raising the possibility that the released domain may translocate to the nucleus to regulate gene expression, in analogy to the Notch signalling pathway. Using full length cadherins fused to either Gal4 or Gal4VP16 transcription factors in combination with Gal4-dependent reporter assays, it was shown that cadherins translocate to the nucleus, but are unable to function as transcriptional activators. Interestingly, nuclear translocation appeared to be independent of PS1 and 2. Together, the presented results suggest that proteolytic cleavage not only alters cadherin adhesive activity, but may also serve as a mechanism to signal changes in adhesiveness to the nucleus

Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification by the Wnt/Beta-Catenin Pathway

Following fertilization in many multicellular organisms, zygotic transcription is suppressed for several hours and cell divisions, until a major embryonic transition termed the midblastula transition (MBT). Nevertheless, steps critical for later patterning of the embryo occur during this early stage. To address this question, we have optimized the chromatin immunoprecipitation technique to allow the investigation of pre-MBT chromatin architecture. We find that, in the context of transcriptional quiescence before the MBT in Xenopus, Wnt signaling through β-catenin primes dorsal gene expression by establishing transcriptionally poised chromatin architecture at target promoters. This is later resolved into active gene expression following the large-scale activation of zygotic transcription at the MBT. During pre-MBT dorsal specification, β-catenin interacts with a histone H3 methyltransferase activity that targets arginine 8 (R8). Recruitment of the arginine methyltransferase Prmt2 to β-catenin target promoters is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3R8 methylation plays a critical role downstream of β-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression. This work demonstrates a mechanism whereby a signal transduction pathway can establish poised chromatin architecture at target genes, which could have implications for the regulation of gene regulatory networks during development. Additionally, our results suggest the possibility that transcriptional poising plays a broader role in maintaining zygotic genome silencing before the MBT